African trypanosomiasis has two major manifestations: the human disease known as Sleeping Sickness and the animal disease Nagana (meaning loss of spirits in the Zulu language). Animal trypanosomiasis is endemic in equatorial Africa, where it is transmitted, among humans and animals, by several species of Glossina, the Tsetse fly. The native African fauna are almost universally infected, providing a vast reservoir of potential human- infective pathogens. Focal human epidemics are a serious reality and an increasing threat. If untreated, trypanosomiasis is rapidly fatal and is currently estimated to kill around 100,000 people annually, although these estimates are compromised by the civil unrest and lack of efficient medical and surveillance programs in much of the affected territories. The few available drugs are old, highly unsatisfactory, acutely toxic and often lethal. The major reason for the persistence of African trypanosomes is their unique and highly efficient mechanism to evade the mammalian immune response. Each trypanosome cell in the infective population shields its surface membrane with a replaceable molecular `coat'composed of 10 million molecules of a single species of Variant Surface Glycoprotein (VSG). In a process known as Antigenic Variation, the majority of the trypanosome population is destroyed by the immune responses to the VSG coat, but individual trypanosomes switch their VSG, using a repertoire of hundreds of VSG genes, evade destruction, and seed successive waves of infection. By developing methods for culturing and genetically manipulating trypanosomes in vitro, we have moved the study of antigenic variation from the descriptive to the analytical. To be expressed, a VSG gene has to be located in a polycistronic telomeric Expression Site (ES), but this is not sufficient for transcription: there may be as many as 20 potential ESs, but only one is active at any time. VSG switching can occur either by shuttling previously silent VSG genes through the currently active ES, or by switching between silent and active ESs. The focus of this proposal is to identify mechanisms that regulate ES transcription, with special emphasis on the role of chromatin structure and histone modifications, and to develop improved genetic techniques for elucidating the mechanisms underlying antigenic variation. The most persuasive current model for the regulation of ES transcription is that it involves histone modifications that affect the overall organization of the nucleosome. The characterization of such modifications, for the first time in trypanosomes, constitutes a major part of our ongoing and proposed experiments. It has been proposed that the ES has to be at a specific sub-nuclear location, dubbed the ES Body, in order to be transcribed. It is likely that access to this site is regulated by histone modifications. We will also use transposon mutagenesis as a method for 'forward'genetics, for identifying the novel genes that are undoubtedly involved in ES silencing and switching, both in the bloodstream and during the developmental cycle as trypanosomes pass through the tsetse/Human trypanosomiasis (also known as Sleeping Sickness) is endemic in equatorial Africa and appears to have returned to levels not seen since the early part of the 20th century. Animal trypanosomiasis also remains a significant problem, not only because it is the major source of human infection with the virulent Trypanosoma brucei rhodesiense sub-species. The primary property that makes these nucleated unicellular organisms so lethal, as they invariably are, is that they can evade our immune defenses by a process of antigenic variation that is far more extensive and effective than the variation employed by some other human pathogens, including HIV and influenza viruses. This project aims to understand how antigenic variation works, at the molecular level, in the African trypanosomes. Such understanding will identify points at which antigenic variation might be disrupted, generating targets for developing urgently needed trypanocidal drugs. Antigenic variation is also a major example of the wider genetic phenomenon of allelic exclusion the expression of one among many essentially identical genetic loci that is a feature of several major gene families in mammals. Trypanosomes, which are relatively easy to study in the laboratory, provide an interesting and experimentally accessible model system in which to explore the mechanism of this more general phenomenon of allelic exclusion. Trypanosomes have many features in common with the cells of higher organisms, including ourselves, and provide an interesting and simplified model to help understand how the more complex genetic regulatory mechanisms of mammals have evolved.